豇豆病毒病病原的分子鉴定

为澄清浙江省豇豆病毒病病原及其分类,采用马铃薯Y病毒属通用引物Sprimer扩增了浙江豇豆线状病毒基因组3′－末端序列。同时又根据已知CMV RNA3序列设计引物pCMV-44-63/1200-1181,扩增了混合的CMV－ZJ运动蛋白基因全序列。外壳蛋白氨基酸序列分析表明,产中仅存在一种线状病毒（CV－ZJ）,该病毒与一泰国豇豆蚜传花叶病毒（CABMV）具有最高的同源性（98.6%）,与花生条纹病毒（PStV）、石斛花叶病毒（DeMV0．）、赤豆花叶病毒（AZMV）、黑眼豆豆花叶病毒（BICMV）和菜豆普通花叶病毒（BCMV）分离物间的同源性为88.5%－94.4%,而与其它CABMV分离物的同源性均低于70％。进化树分析表明,PStV、AZMV、DeMV、BlCMV和BCMV为同种异名,应统称为BCMV,而CABMV为另一种病毒;CV－ZJ和泰国CABMV分离物应分类为BCMV豇豆株系。CMV－ZJ的运动蛋白序列分析表明,该分离物属于CMV亚组Ⅰ,与其它分离物氨基酸序列同源性为93.1%－97.8%。     

A fluorescent PCR assay for the codominant interpretation of a dominant SCAR marker linked to the virus resistance allele bc-1 2 in common bean

Our objective was to develop a rapid and accurate procedure to genotype common bean plants for the bc-1 ² allele, which conditions resistance to bean common mosaic and bean common mosaic necrosis viruses. A segregating F₂ population was derived from the cross between pinto bean breeding lines P94207-43 (bc-1 ²//bc-1 ²) and P94207-189 (bc-1//bc-1). A quantitative PCR assay based on the detection of fluorescent labeled amplicons was developed to distinguish between homozygous (bc-1 ²//bc-1 ²), heterozygous (bc-1 ²//bc-1) and null (bc-1//bc-1) F₂ genotypes. Remnant F₁ plants were used as a comparative reference sample. PCR results among this sample fit a normal distribution, and 99% and 95% confidence intervals for heterozygotes were determined. F₂ plants for which no amplification was detected were classified as null (bc-1//bc-1) genotypes. F₂ plants that fell within the confidence intervals for heterozygotes were classified as heterozygotes (bc-1 ²//bc-1), while plants that fell outside the right tail of the heterozygote confidence intervals were classified as homozygotes (bc-1 ²//bc-1 ²). F₂ plants were also genotyped for the bc-1 ² allele by performing F₃ family progeny tests for virus resistance. Agreement between the two methods for genotyping plants was 100% (59/59) when PCR genotyping was based on a 99% heterozygote confidence interval, and 98.3% (58/59) when based on a 95% heterozygote confidence interval. This assay will accelerate breeding for virus resistance in bean by facilitating discrimination among plants that are heterozygous or homozygous for the bc-1 ² allele. The experimental design may be generally applicable towards developing other assays for the codominant interpretation of dominant markers in diploid plants.     

Serological and molecular properties of isolates of Bean common mosaic virus-BCMV from Khorasan Razavi province

During growing season, 2011–2012, selected bean plant samples with symptoms of mosaic, vein clearing, leaf rolling, were collected from different part of Khorasan Razavi province of north-eastern Iran. To identify the virus, leaf samples were tested serologically by DAS-ELISA and tissue blot, using specific BCMV polyclonal antibody. Elisa positive samples were rechecked by RT-PCR and IC-RT-PCR using set of primers directed to the coat protein gene which were designed to detect and characterise the viral species. Similarly, the BCMV primers amplified product of approximately 373?bp in 1% agarose gel electrophoresis. In phylogenic analysis, A and B groups were formed for 12 Iranian isolate comparing with 18 BCMV isolates from GenBank. Iranian isolates were classified into three clusters indicating to have more homology 99.3% with Mexican Isolates. There are also indications of some diversities among Iranian BCMV isolates.     

Immunocapture RT-PCR detection of Bean common mosaic virus and strain blackeye cowpea mosaic in common bean and black gram in India

The strains of Bean common mosaic virus (BCMV) and blackeye cowpea mosaic (BICM), genus Potyvirus, were detected from 25 common bean and 14 black gram seeds among 142 seed samples collected from different legume-growing regions of India. The samples were subjected to a growing-on test, an indicator plant test, an electron microscopic observations, an enzyme linked immunosorbent assay and an immunocapture RT-PCR. The incidence of the two tested viruses in common bean and black gram seed samples was 1–6% and 0.5–3.5%, respectively in growing-on test evaluations. Electron microscopic observations revealed filamentous virion particles from the leaves of plants showing characteristic virus disease symptoms in growing-on and host inoculation tests. The identity of the strains was confirmed by immunocapture RT-PCR, with a final amplification product of approximately 700 bp for BCMV and BCMV–BICM. The complete identity of the two viruses was further confirmed by nucleotide sequencing of the partial coat protein and 3′-UTR regions. The sequences of the four BCMV and BCMV–BICM isolates each consisted of 583–622 and 550–577 nucleotides. The present report confirms the widespread nature of these two serious potyviruses in the two most important legume crops in India.     

Occurrence of latent virus infection in visually-rated cowpea (Vigna unguiculata L. Walp) seedlings

Abstract

The presence of latent infections was studied in five cowpeas varieties. Seeds of the varieties were planted and the seedlings inoculated with antigens from Cucumber mosaic virus (CMV) genus Cucumovirus, Bean common mosaic virus (BCMV) genus Potyvirus (Blackeye cowpea mosaic virus strain), Southern bean mosaic virus (SBMV) genus Sobemovirus and Cowpea mottle virus (CPMoV) genus Carmovirus seven days after planting. Seedlings expressing symptoms were rouged at two weeks after inoculation, while asymptomatic ones were subjected to serological indexing to detect the presence/absence of latent infection. Protein A-sandwich enzyme-linked immunosorbent assay (PAS ELISA) was employed for the serological detection of CMV, SBMV and CPMoV, while antigen-coated plate (ACP) ELISA was used to detect BCMV in the asymptomatic plants. Cowpea seedlings without virus symptoms but with positive serological reactions were considered as being latently infected. All of the inoculated TVu 1272 and SuVita-2 plants showed symptoms consistent with CMV and CPMoV infections, respectively. The rate of CMV latent infection was high in TVu 1179 (14.5%), low in SuVita-2 (1.3%) but not recorded in TVu 1272.     

Incidence and severity of bean common mosaic disease and resistance of popular bean cultivars to the disease in western Kenya

The common bean (Phaseolus vulgaris) is a high protein crop and the main legume in the cropping system of western Kenya. Despite its importance, common bean yields are low (<1.0 t/ha) and declining. Bean common mosaic virus (BCMV) and bean common mosaic necrosis virus (BCMNV) are the most common and most destructive viruses and can cause a yield loss as high as 100%. In Kenya, a limited number of cultivars and exotic genotypes with resistance to BCMV and BCMNV strains have been reported. This study sought to determine the distribution and screen popular cultivars for resistance to the viruses. In October 2016 and May 2017, two diagnostic surveys for bean common mosaic disease (BCMD) were conducted in seven counties of western Kenya namely Bungoma, Busia, Homa Bay, Nandi, Vihiga, Kakamega and Siaya. Leaf samples showing virus-like symptoms were collected and analysed by ELISA. Sixteen popularly grown bean cultivars together with cowpea (Vigna unguiculata), soybean (Glycine max), green grams (Vigna radiata) and groundnut (Arachis hypogaea) were planted in a greenhouse in a completely randomized block design with three replicates. The plants were inoculated with BCMNV isolate at 3-leaf stage. Data were taken weekly for 3 weeks on type of symptoms expressed and number of plants infected. In total, 270 bean farms were visited. Symptoms of mosaic, downward curling, local lesions, stunting or a combination of these were observed during both surveys. Mean virus incidence was higher in the short rain season (50.2%) than in the long rain season (35.6%). The mean BCMD severity on a scale of 0–3 was highest (2.3) in Kakamega County and lowest (0.5) in Siaya. On variety resistance tests to BCMNV isolate, 10 bean cultivars were susceptible, four tolerant and two resistant. BCMNV is widely distributed across counties probably because of use of uncertified seeds by farmers and inoculum pressure from seed and aphid vector. For improved yields of common bean, farmers should be advised to plant certified seeds for all legumes in the cropping system.     

Inheritance of resistance to potyviruses in Phaseolus vulgaris L. III. Cosegregation of phenotypically similar dominant responses to nine potyviruses

We have identified monogenic dominant resistance to azuki bean mosaic poty virus (AzMV), passionfruit woodiness potyvirus-K (PWV-K), zucchini yellow mosaic potyvirus (ZYMV), and a dominant factor that conditioned lethal necrosis to Thailand Passiflora potyvirus (ThPV), in Phaseolus vulgaris Black Turtle Soup 1. Resistance to AzMV, PWV-K, ZYMV, watermelon mosaic potyvirus, cowpea aphid-borne mosaic potyvirus, blackeye cowpea mosaic potyvirus, and lethal necrosis to soybean mosaic potyvirus and ThPV cosegregated as a unit with the I gene for resistance to bean common mosaic potyvirus.     

感染菜豆普通花叶病毒植株的形态、生理代谢及产量性状与病毒浓度的关系

在四个菜豆品种“沙克沙”、“家雀蛋”、“自来油”、“白大架”上按种菜豆花叶病毒(Bean Common Mosaic Virus(BCMV)V-5],接种后15—25天测定,病毒在植株体内的浓度达到高峰。同时,植高、叶面积,叶绿素总量,光合速率较健康株明显下降,呼吸强度、游离态氨基酸总量上升。接种后30—35天,病毒含量开始下降,光合速率,呼吸强度、株高几乎接近正常水平。叶面积、叶绿素总量,游离态氨基酸总量同健株的差异也大大减少。继病毒侵入后植株生理代谢的失调,温室盆栽条件下,菜豆不同品种的产量损失为40—64％。     

Serological and molecular characterisations of the Egyptian isolate of Bean common mosaic virus

Bean common mosaic virus (BCMV) was isolated from the naturally infected bean plants collected from the Kafr El-Sheikh and El-Gharbia Governorates. BCMV induced sever mosaic, vein banding, malformation, leaf curling and stunting on bean plants cv. Giza 6. The isolated virus was propagated in bean plants cv. Giza 6. The identification of BCMV was carried out serologically by an indirect enzyme-linked immunosorbent assay using BCMV antiserum. Positive reaction indicated that the virus under study was related serologically to Potyvirus. The molecular biology techniques were used to identify and characterise the coat protein gene of BCMV. Oligonucleotide primers were designed for BCMV according to the published nucleotide sequences of BCMV and were successfully amplified with a DNA fragment (300 bp) from BCMV CP gene by RT-PCR. The total RNA was extracted from bean leaves and was reverse-transcribed and amplified using the oligonucleotide primer. The amplified product was analysed by gel electrophoresis. Also, Southern and dot blot hybridisations were used to establish the authenticity and specificity to the RT-PCR-amplified products of BCMV. The nucleotide sequences of the Egyptian isolate of BCMV/CP showed similarity with an isolate (BCMV-NY 15) which belongs to Puerto Rico.